Enzyme inhibitors

ABSTRACT

Compounds of general formula (I):  
                 
 
     where A, E, G, X, Y and the bond - - - take various meanings are of use in the preparation of a pharmaceutical formulation, for example in the treatment of a disease in which GSK-3 is involved, including Alzheimer&#39;s disease or the non-dependent insulin diabetes mellitus, or hyperproliferative disease such as cancer, displasias or metaplasias of tissue, psoriasis, arteriosclerosis or restenosis.

FIELD OF THE INVENTION

[0001] The present invention relates to enzyme inhibitors, and moreparticularly to heterocyclic inhibitors of glycogen synthase kinase 3β,GSK-3.

BACKGROUND OF THE INVENTION

[0002] Alzheimer's disease (AD) is a neurodegenerative processcharacterised by cognitive disorders associated with a progressivedeterioration of the cholinergic function, and neuropathological lesionsas senile plaques, formed by the fibrillary β-amyloid, andneurofibrillary tangles, bundles of paired helical filaments.

[0003] Generally speaking, AD is restricted to groups aged 60 years ormore and is the most common cause of dementia in the elderly population.Today, AD affects 23 million people worldwide. As longevity increases,it is estimated that by the year 2050 the number of cases of AD willmore than triplicate [Amaduci, L.; Fratiglioni, L. “Epidemiology of AD:Impact on the treatment”, in Alzheimer Disease: Therapeutic Strategies,E. Giacobini and R. Becker, Eds., Birhauser, EEUU, 1994, pp. 8].

[0004] Two major histological lesions are observed in AD brainsassociated with the neuronal loss: neurofibrillary tangles and senileplaques at the intracellular and extracellular level respectively[“Alzheimer Disease: From molecular biology to therapy”, E. Giacobiniand R. Becker, Eds., Birhauser, EEUU, 1996].

[0005] Neurofibrillary tangles are structures formed by paired helicalfilaments (PHFs). They are comprised mainly of themicrotubule-associated protein (MAP) tau in an abnormallyhyperphosphorylated state [Grundke-Iqbal, I.; Iqbal, K.; Tung, Y. C.;Quinlan, M.;

[0006] Wisniewski, H. M.; Binder, L. I., “Abnormal phosphorylation ofthe microtubule-associated protein tau in Alzheimer cytoskeletalpathology”, Proc. Natl. Acad. Sci. USA, 1986, 83, 4913-4917;Grundke-Iqbal, I.; Iqbal, K.; Quinlan, M.; Tung, Y. C.; Zaidi, M. S.;Wisniewski, H. M., “Microtubule-associated protein tau. A component ofthe Alzheimer paired helical filaments”, J. Biol. Chem., 1986, 261,6084-6089; Greenberg, S. G.; Davies, P.; Schein, J. D.; Binder, L. I.,“Hydrofluoric acid-treated tau PIIF proteins display the samebiochemical properties as normal tau.”, J. Biol. Chem., 1992, 267,564-569]. Such aberrant phosphorylation of tau, determined by theeffects of different protein kinases and phosphatases, appears tocompromise on its ability to bind to and stabilise microtubules and thismay contributes to AD pathology [Moreno, F. J.; Medina, M.; Perez, M.;Montejo de Garcini, E.; Avila, J., “Glycogen sintase kinase 3phosphorylation of different residues in the presence of differentfactors: Analysis on tau protein”, FEBS Lett., 1995, 372, 65-68]. Thus,the blockade of this hyperphosphorylation step may be a prime target atwhich to interrupt the pathogenic cascade. The selective inhibitors oftau kinases might be new effective drugs for the treatment of AD.

[0007] The search for tau kinases inhibitors is a field of a greatinterest. Tau can be phosphorylated by several proline-directed proteinkinases (PDKs) and non-PDKs. However, in AD the exact role of any ofthese kinases in the abnormal hyperphosphorylation of tau is not yetunderstood and to date, the activity of these kinases has not been foundto be upregulated. It is no doubt that glycogen synthase kinase 3,(GSK-3β) is an in vivo tau kinase in the brain [Lovestone, S.; Hartley,C. L.; Pearce, J.; Anderton, B. H., “Phosphorylation of tau by glycogensynthase-3 in intact mammalian cells: the effects on the organizationand stability of microtubules”, Neuroscience, 1996, 73, 1145-1157;Wagner, U.; Utton, M.; Gallo, J. M.; Miller, C. C., “Cellularphosphorylation of tau by GSK-3β influences tau binding to microtubulesand microtubule organisation”, J. Cell. Sci., 1996, 109, 1537-1543;Ledesma, M.; Moreno, F. J.; Perez, M. M.; Avila, J., “Binding ofapolipoprotein E3 to tau protein: effects on tau glycation, tauphosphorylation and tau-microtubule binding, in vitro”, Alzheimer Res.,1996, 2, 85-88]. These findings open the gate to the use of GSK-3,inhibitors as therapeutical agents in the treatment of AD. At the momentfew compounds are known with this enzymatic inhibitory property.

[0008] Lithium behaves as a specific inhibitor of the GSK-3 family ofprotein kinases in vitro and in intact cells Muñoz-Montanfo, J. R.;Moreno, F. J.; Avila, J.; Diaz-Nido, J., “Lithium inhibits Alzheimer'sdisease-like tau protein phosphorylation in neurons”, FEBS Lett., 1997,411, 183-188].

[0009] Finally, it is observed that insulin inactivates GSK-3 and it isshown that the non-dependent insulin diabetes mellitus is developed withthe activation of this enzyme. So that, GSK-3 inhibitors would be a newtherapy for the non-dependent insulin diabetes mellitus.

[0010] In our work team we have recently discovered a new family ofsmall synthetic heterocyclic molecules with GSK-30 inhibitory propertiesat micromolar level.

DESCRIPTION OF THE INVENTION

[0011] The invention is directed to the compounds represented by thegeneral formula I:

[0012] where:

[0013] A is —C(R¹)₂—, —O— or —NR¹—;

[0014] E is —NR¹— or —CR¹R²— and the substituent R² is absent if - - -is a second bond between E and G;

[0015] G is —S—, —NR¹— or —CR¹R²— and the substituent R² is absentif - - - is a second bond between E and G;

[0016] - - - may be a second bond between E and G where the nature of Eand G permits and E with G optionally then forms a fused aryl group;

[0017] R¹ and R² are independently selected from hydrogen, alkyl,cycloalkyl, haloalkyl, aryl, —(Z)_(n)-aryl, heteroaryl, —OR³, —C(O)R³,—C(O)OR³, —(Z)_(n)—C(O)OR³ and —S(O)_(t)— or as indicated R can be suchthat E with G then form a fused aryl group;

[0018] Z is independently selected from —C(R³)(R⁴)—, —C(O)—, —O—,—C(═NR³)—, —S(O)_(t)—, N(R³)—;

[0019] n is zero, one or two;

[0020] t is zero, one or two;

[0021] R³ and R⁴ are independently selected from hydrogen, alkyl, aryland heterocyclic; and

[0022] X and Y are independently selected from ═O, ═S, ═N(R³) and═C(R¹)(R²).

DETAILED DESCRIPTION OF THE INVENTION

[0023] As used in this specification and appended claims, unlessspecified to the contrary, the following terms have the meaningindicated:

[0024] “Alkyl” refers to a straight or branched hydrocarbon chainradical consisting of carbon and hydrogen atoms, containing nosaturation, having one to eight carbon atoms, and which is attached tothe rest of the molecule by a single bond, e.g., methyl, ethyl,n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, etc. Alkyl radicals maybe optionally substituted by one or more substituents independentlyselected from the group consisting of a halo, hydroxy, alkoxy, carboxy,cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro, mercapto andalkylthio.

[0025] “Alkoxy” refers to a radical of the formula —OR_(a) where R_(a)is an alkyl radical as defined above, e.g., methoxy, ethoxy, propoxy,etc.

[0026] “Alkoxycarbonyl” refers to a radical of the formula —C(O)OR_(a)where R_(a) is an alkyl radical as defined above, e.g., methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, etc.

[0027] “Alkylthio” refers to a radical of the formula —SR_(a) whereR_(a) is an alkyl radical as defined above, e.g., methylthio, ethylthio,propylthio, etc.

[0028] “Amino” refers to a radical of the formula —NH₂.

[0029] “Aryl” refers to a phenyl or naphthyl radical, preferably aphenyl radical. The aryl radical may be optionally substituted by one ormore substituents selected from the group consisting of hydroxy,mercapto, halo, alkyl, phenyl, alkoxy, haloalkyl, nitro, cyano,dialkylamino, aminoalkyl, acyl and alkoxycarbonyl, as defined herein.

[0030] “Aralkyl” refers to an aryl group linked to an alkyl group.Preferred examples include benzyl and phenethyl.

[0031] “Acyl” refers to a radical of the formula —C(O)—R_(c) and—C(O)—R_(d) where R_(c) is an alkyl radical as defined above and R_(d)is an aryl radical as defined above, e.g., acetyl, propionyl, benzoyl,and the like.

[0032] “Aroylalkyl” refers to an alkyl group substituted with—C(O)—R_(d). Preferred examples include benzoylmethyl.

[0033] “Carboxy” refers to a radical of the formula —C(O)OH.

[0034] “Cyano” refers to a radical of the formula —CN

[0035] “Cycloalkyl” refers to a stable 3- to 10-membered monocyclic orbicyclic radical which is saturated or partially saturated, and whichconsist solely of carbon and hydrogen atoms. Unless otherwise statedspecifically in the specification, the term “cycloalkyl” is meant toinclude cycloalkyl radicals which are optionally substituted by one ormore substituents independently selected from the group consisting ofalkyl, halo, hydroxy, amino, cyano, nitro, alkoxy, carboxy andalkoxycarbonyl.

[0036] “Fused aryl” refers to an aryl group, especially a phenyl orheteroaryl group, fused to the five-membered ring.

[0037] “Halo” refers to bromo, chloro, iodo or fluoro.

[0038] “Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, and the like.

[0039] “Heterocycle” refers to a heterocyclyl radical. The heterocyclerefers to a stable 3- to 15-membered ring which consists of carbon atomsand from one to five heteroatoms selected from the group consisting ofnitrogen, oxygen, and sulfur, preferably a 4- to 8-membered ring withone or more heteroatoms, more preferably a 5- or 6-membered ring withone or more heteroatoms. For the purposes of this invention, theheterocycle may be a monocyclic, bicyclic or tricyclic ring system,which may include fused ring systems; and the nitrogen, carbon or sulfuratoms in the heterocyclyl radical may be optionally oxidised; thenitrogen atom may be optionally quaternized; and the heterocyclylradical may be partially or fully saturated or aromatic. Examples ofsuch heterocycles include, but are not limited to, azepines,benzimidazole, benzothiazole, furan, isothiazole, imidazole, indole,piperidine, piperazine, purine, quinoline, thiadiazole, tetrahydrofuran.The hetrocycle may be optionally substituted by R³ and R⁴ as definedabove in the summary of the invention.

[0040] “Heteroaryl” refers to an aromatic heterocycle

[0041] “Mercapto”refers to a radical of the formula —SH

[0042] “Nitro” refers to a radical of the formula —NO₂

[0043] The invention is in particular directed to the enzymatic activityagainst kinases of the compounds of the general formula I.

[0044] A is preferably selected from —C(R¹)₂— and —NR¹—.

[0045] Preferably R¹ is selected from hydrogen, alkyl, cycloalkyl, aryl(optionally substituted with a group selected from alkyl, halo andalkoxy), —C(R³)(R⁴)-aryl (the aryl part being optionally substitutedwith a group selected from alkyl, halo and alkoxy), —OR³, —C(O)OR³ and

[0046] —C(R³)(R⁴)—C(O)OR³, and R³ and R⁴ are independently selected fromhydrogen and alkyl.

[0047] The subscript n is preferably zero or one, and n will be chosenhaving regard to the known chemistry of possible groupings.

[0048] X and Y are preferably oxygen or sulphur, at least one of X and Yis preferably oxygen.

[0049] A particularly preferred class of compounds is of the formula(II).

[0050] where R_(a) and R_(b) are independently selected from hydrogen,alkyl, cycloalkyl, haloalkyl, aryl,

[0051] —(Z)_(n)-aryl, heteroaryl, —OR³, —C(O)R³, —C(O)OR³,—(Z)_(n)—C(O)OR³ and —S(O)_(t)—, and

[0052] Z, n, t, R³, R⁴, X and Y are as defined above.

[0053] In the formula (II), X and Y are preferably selected from oxygen,sulphur, and —NR³— where R³ is heterocyclic, especially a 6-memberedheterocycle which has one heteroatom which is nitrogen, being optionallyaromatic and optionally oxidised or quaternised. More preferably, both Xand Y are both oxygen.

[0054] Preferably. R^(a) and R^(b) are independently selected fromhydrogen, alkyl, cycloalkyl, aryl (optionally substituted with a groupselected from alkyl, halo and alkoxy), —C(R³)(R⁴)-aryl (the aryl partbeing optionally substituted with a group selected from alkyl, halo andalkoxy),

[0055] —OR³, —C(O)OR³ and —C(R³)(R⁴)—C(O)OR³, and R³ and R⁴ areindependently selected from hydrogen, alkyl and heterocyclic.

[0056] More preferably R_(a) and R_(b) are independently selected fromalkyl, aryl (optionally substituted with a group selected from alkyl,halo and alkoxy), —CH₂-aryl (the aryl part being optionally substitutedwith a group selected from alkyl, halo and alkoxy), and —CH₂—C(O)OR³where R³ is hydrogen or alkyl.

[0057] Still more preferably, R^(a) and R^(b) are independently selectedfrom methyl, ethyl, propyl, benzyl, phenyl (optionally substituted witha group selected from methyl, fluoro, chloro, bromo and methoxy) and—CH₂—C(O)O-ethyl.

[0058] The most preferred compounds of formula (II) are listed in TableI below. TABLE 1 R^(a) R^(b) X Y CH₂Ph Me O O Et Me O O Ph Me O OCH₂CO₂Et Me O O 4-OMePh Me O O 4-MePh Me O O 4-BrPh Me O O 4-FPh Me O O4-ClPh Me O O CH₂Ph CH₂Ph O S Ph Ph O S

[0059] Another preferred class of compounds of the invention are thosecompounds of formula (III):

[0060] wherein:

[0061] B is —NR⁷— or C(R⁷)(R⁸)-(wherein R⁷ and R⁸ are independentlyselected from hydrogen,

[0062] alkyl, aryl, —CH₂—W-aryl, and —W—CO₂H, and W is a single bond,CH₂ or CO);

[0063] R⁵ and R⁶ are independently selected from hydrogen, alkyl, aryland —CH₂-aryl; and

[0064] X and Y are independently selected from ═O and ═S.

[0065] In the formula (III), B is preferably —NR⁷—, wherein R⁷ isselected from hydrogen, alkyl and —CH₂-aryl, especially hydrogen, methylor benzyl.

[0066] R⁵ and R⁶ are preferably hydrogen.

[0067] X and Y are preferably oxygen.

[0068] The most preferred compounds of formula (III) are listed in Table2 below. TABLE 2 B X Y R⁵ R⁶ NH O O H H N-CH₂Ph O O H H NMe O O H H CH₂O O H H

[0069] Examples of further classes of compounds of formula I includethose where:

[0070] a) A is —CH₂—; E is —CR¹R²—, preferably —CH₂—; G is —CR¹R²—,preferably —CH₂—;

[0071] b) A is —CH₂—; E is —CR¹—, preferably —CH—; G is —CRY—,preferably —CH—; and - - - is a second bond between G and E;

[0072] c) A is —O—; E is —CR¹—, preferably —CH—; G is —CRY—, preferably—CH—; and - - - is a second bond between G and E;

[0073] d) A is —NR¹—, where R¹ is preferably hydrogen, alkyl or aralkyl;E is —CR¹—, preferably —CH—; G is —CR¹—, preferably —CH—; and - - - is asecond bond between G and E;

[0074] e) A is —NR¹—, where R¹ is preferably hydrogen or aralkyl; E is—CR¹R —, preferably —CH₂—; G is —CR¹R²—, preferably —CH₂—;

[0075] f) A is —NR¹—, where R¹ is preferably hydrogen or aralkyl; E is—CR¹—; G is —CR¹—; - - - is a second bond between E and G; and E with Gform a fused aryl group, preferably a phenyl group;

[0076] g) A is —NR¹—, where R¹ is preferably hydrogen, alkyl,carboxyalkyl, aroylalkyl or aralkyl; E is —S; G is —C(R¹)₂—, preferably—CH₂—;

[0077] h) A is —NR¹, where R¹ is preferably aryl; E is —R¹—, where R¹ ispreferably hydrogen or alkyl; G is —NR¹—, where R¹ is preferablyhydrogen or alkyl.

[0078] In these classes of compounds, X and Y are preferably both O,though for class (g) X can be O and Y can be S. When E with G form afused phenyl group, the resultant compounds are phthalimido derivatives.

SYNTHESIS OF THE COMPOUNDS OF THE INVENTION

[0079] The compounds of the invention can be synthesised by availableprocedures.

[0080] For preferred compounds of formula (II) a general procedure isavailable [Martinez, A.; Castro, A.; Cardelús, I.; Llenas, J.; Palacios,J. M. Bioorg. Med. Chem., 1997, 5, 1275-1283].

[0081] Concretely, the compounds of general formula (II) and collectedin Table I, were prepared following the synthetic procedure depicted inscheme 1, and using the reactivity ofN-alkyl-S-[N′-chlorocarbamoyl)amino]isothiocarbamoyl chlorides withdifferent alkyl isocyanates. The isothiocyanates chlorination isperformed by addition of an equimolecular quantity of chlorine over anhexane solution of the mentioned isothiocyanate at −15° C. The reactionof the iminochloroalkylsulfenyl chloride formed with alkyl or arylisocyanate under inert atmosphere and subsequent hydrolysis, yielded thethiadiazolidinediones described in table I.

[0082] The typical compounds of this invention selectively inhibitGSK-3β without inhibition of others protein kinases such as PKA, PKC,CK-2 and CdK2, which could eliminate the widespread effects. GSK-3β isinvolved in the aetiopathogenesis of AD and it is responsible for theabnormal hyperphosphorylation of the tau protein. The selectiveinhibitors here disclosed can be useful therapeutical agents for thetreatment of neurodegeneratives diseases associated to the pathology oftau protein, specially for AD which forms part of this invention. Theinhibitory action of these compounds against GSK-3, leads for the designof drugs able to stop the formation of the neurofibrilar tangles, one ofthe hallmark present in this neurodegenerative process.

[0083] These compounds can be useful for the treatment of otherpathologies in which the GSK-3β is involved, such asnon-insulin-dependent diabetes mellitus.

[0084] Additionally, these compounds can be useful for the treatment ofhyperproliferative diseases such as displasias and metaplasias ofdifferent tissues, psoriasis, artherioschlerosis, resthenosis andcancer, due to their inhibition of cellular cycle which forms part ofthis invention.

[0085] Accordingly, the present invention further providespharmaceutical compositions comprising a compound of this inventiontogether with a pharmaceutically acceptable carrier or diluent.Appropriate dosage forms and dosing rates can be devised and adopted inaccordance with conventional practice.

EXAMPLE Example 1 Enzymatic Inhibition of the Compounds of the Invention

[0086] GSK-3β inhibition: The GSK-3 activity was determined byincubation of a mixture of GSK-3 enzyme (Sigma), a phosphate source anda GSK-3 substrate in the presence and in the absence of thecorresponding test compound, and by measuring the GSK-3 activity of thismixture.

[0087] Concretely, the GSK-3 activity is determined by incubating theenzyme at 37° C. during 20 minutes in a final volume of 12 μl of buffer(50 mM tris, pH=7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 mM Cl₂Mg)supplemented with 15 μM (final concentration) of the synthetic peptideGS 1 [Woodgett, J. R. “Use of peptides for affinity purification ofprotein-serine kinases”, Anal. Biochem., 1989, 180, 237-241] assubstrate, 15 μM of ATP, 0.2 μl of [γ-³²P]ATP and differentconcentrations of the test compound. The reaction is quenched byaddition of an aliquot of the reaction mixture in phosphocelullose p81papers. These papers are washed three times with phosphoric acid 1% andthe radioactivity incorporated to the GS 1 peptide is measured in aliquid scintillation counter.

[0088] Compounds showed in table 1 are representative of the GSK-3inhibitory activity object of this invention. The IC₅₀ (concentration atwhich a 50% of enzyme inhibition is shown) values are gathered in Table3 below. The synthesis of the compounds listed in Table 3 is describedbelow. TABLE 3 (II)

Compound IC₅₀ No. R^(a) R^(b) X Y (μM) 1 CH₂Ph Me O O 1 2 Et Me O O 5 3Et nPr O O 10 4 Et cyclo- O O 10 hexyl 5 Ph Me O O 2 6 CH₂CO₂Et Me O O 57 4-OMePh Me O O 5 8 CH₂Ph Et O O 7 9 Et iPr O O 35 10 CH₂Ph Et O S 6 11CH₂Ph CH₂Ph O S 10 12 Ph Ph O S 20 13 Et Et O S 20 14 Cyclohexyl Me OO >100 15 4-MePh Me O O 5 16 4-BrPh Me O O 3 17 4-FPh Me O O 4 18 4-ClPhMe O O 4 19 Et Me

O >100 20 Et Et

O >100 21 Et H

O >100 22 Me Me

O >100 23 Et Me

O >100 24 Et Me

O >100 25 Et Me

O >100 26 Et Me

S 10

Methods for the Synthesis of the Compounds Depicted in Table 3

[0089] General Method for the Synthesis of1,2,4-Thiadiazolidin-3,5-diones (Compounds 1-18)

[0090] Chlorine (generated by the addition of 35% HCl to KMnO₄) wasbubbled slowly through a solution of aryl or alkyl isothiocyanate in dryhexane (25 ml), under a nitrogen atmosphere, at −15° C. to −10° C. Thetemperature of the reaction mixture was carefully controlled during theaddition step. At this point, the N-aryl orN-alkyl-S-chloroisothiocarbamoyl chloride (see Scheme 1 above) wasformed. Afterwards, alkyl isocyanate was added, and the mixture wasstirred at room temperature for between 8 and 10 h. After this time, theresulting product was purified by suction filtration andrecrystallization or silica gel column chromatography using theappropriate eluant. Sometimes, the 5-oxo-1,2,4-thiadiazolidine-3-thionewas isolated as a by-product.

[0091] Specific Methods and Data for the Compounds Listed in Table 3

[0092] 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (Compound 1)and 2,4-Dibenzyl-5-oxo-thiadiazolidine-3-thione (compound 11):

[0093] Reagents: Benzyl iso-thiocyanate (0.86 ml, 6.5 mmol), 35% HCl(3.1 ml), KMnO₄ (0.5 g), methyl isocyanate (0.38 ml, 6.5 mmol).

[0094] Conditions: Room temperature, 8 h.

[0095] Isolation (1): filtration of reaction mixture.

[0096] Purification: recrystallization from hexane.

[0097] Yield: 0.75 g (35%) as white solid; mp 60-61° C. ¹H-NMR (CDCl₃):3.2 (s, 3H, CH₃); 4.8 (s, 2H, CH₂-Bn); 7.31-7.45 (m, 5H, arom.) ¹³C-NMR(CDCl₃): 31.4 (CH₃); 46.0 (CH₂-Bn) 128.2; 128.6; 128.8; 135.1 (C arom.);155.2 (3-C═O); 165.6 (5-C═O); Anal. (C₁₀H₁₀N₂SO₂) C, H, N, S.

[0098] Isolation (11): The filtrate was evaporated.

[0099] Purification: silica gel column chromatography using CH₂Cl₂/Hexane (1:1).

[0100] Yield: 0.08 g (8%) as yellow solid; mp 91-95° C. ¹H-NMR (CDCl₃):4.52 (s, 2H, CH₂-Bn); 5.10 (s, 2H, CH₂-Bn); 7.31-7.52 (m, 10H, arom¹³C-NMR (CDCl₃): 50.1 (CH₂-Bn); 54.3 (CH₂-Bn); 128.1; 128.4; 128.9;135.4 (C arom.); 127.1; 127.4; 128.4; 138.6 (′C arom.); 148.1 (3-C═S);169.0 (5-C═O); Anal. (C₁₆H₁₄N₂S₂O) C, H, N, S.

[0101] 4-Ethyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (Compound 2):

[0102] Synthesis of this compound is described in Martinez, A.; Alonso,D.; Castro, A.; Arán, J. V.; Cardelus, I.; Banõs, J. E.; Badía, A.,Arch. Pharm. Pharm. Med. Chem., 1999, 332, 191-194, the contents ofwhich are incorporated herein by reference thereto.

[0103] 4-Ethyl-2-n-propyl-1,2,4-thiadiazolidine-3,5-dione (Compound 3):

[0104] Synthesis of this compound is described in Martinez, A.; Alonso,D.; Castro, A.; Arán, J. V.; Cardelus, I.; Baĩos, J. E.; Badía, A.,Arch. Pharm. Pharin. Med. Chem., 1999, 332, 191-194; the contents ofwhich are incorporated herein by reference thereto.

[0105] 2-Cyclohexyl-4-ethyl-1,2,4-thiadiazolidine-3,5-dione (Compound 4)and 2,4-diethyl-5-oxo-1,2,4-thiadiazolidine-3,5-dione (Compound 13).

[0106] Reagents: Ethyl isothiocyanate (0.56 ml, 6.5 mmol), 35% HCl (3.1ml), KMnO₄ (0.5 g), cyclohexyl isocyanate (0.825 ml, 6.5 mmol).

[0107] Conditions: Room Temperature, 10 h.

[0108] Purification: silica gel column chromatography using AcOEt/Hexane(1:10).

[0109] Yield: The first fraction 0.12 g of compound 4 (2%) as yellowoil. ¹H-NMR (CDCl₃): 1.20 (t, 3H, CH₂CH₃, J=7.1 Hz); 1.31 (t, 3H,CH₂′CH₃, J=7.2 Hz); 3.33 (c, 2H, ′CH₂CH₃, J=7.2 Hz); 3.89 (c, 2H,CH₂CH₃, J=7.1 Hz). ¹³C-NMR (CDCl₃); 12.2 (CH₂CH₃); 15.7 (CH₂′CH₃); 42.4(CH₂CH₃); 45.8 (′CH₂CH₃); 146.3 (3-C═S); 168.2 (5-C═O). Anal.(C₆H₁₀N₂OS₂) C, H, N, S.

[0110] The second fraction 0.73 mg of compound 13 as white solid (49%);mp=45-48° C.

[0111]¹H-NMR (CDCl₃): 1.20 (t, 3H, CH₂CH₃, J=7.1 Hz); 1.31-1.92 (m, 5H,chex); 3.72 (c, 2H, CH₂CH₃, J=7.1 Hz). ¹³C-NMR (CDCl₃): 13.0 (CH₂CH₃);39.8 (CH₂CH₃); 24.7; 25.1; 31.73; 53.71(C chex); 152.2 (3-C═O); 166.2(5-C═O); Anal. (C₁₀H₁₆N₂O₂S) C, H, N, S.

[0112] 4-Phenyl-2-methyl-1,2,4-thiadiazolidin-3,5-dione (Compound 5) and2,4-Diphenyl-5-oxo-thiadiazolidine-3-thione (compound 12).

[0113] Reagents: Phenyl isothiocyanate (0.78 ml, 6.5 mmol), 35% HCl (3.1ml), KMnO₄ (0.5 g), methyl isocyanate (0.38 ml, 6.5 mmol).

[0114] Conditions: Room temperature, 8 h.

[0115] Isolation (5): filtration of reaction mixture.

[0116] Purification: recrystallization from methanol.

[0117] Yield: 0.25 g (30%) as white solid; mp 174-179° C. ¹H-NMR(CDCl₃): 3.21 (s, 3H, CH₃); 7.31-7.50 (m, 5H, arom.). ¹³C-NMR (CDCl₃):31.7 (CH₃); 127.2; 129.2; 129.4; 132.7 (C arom.); 152.7 (3-C═O); 165.3(5-C═O). Anal. (C₈H₈N₂SO₂) C, H, N, S.

[0118] Isolation (12): The filtrate was evaporated.

[0119] Purification: silica gel column chromatography using CH₂Cl₂.

[0120] Yield: 0.14 g (15%) as yellow solid; mp 105-110° C. ¹H-NMR(CDCl₃): 6.70-7.01 (m, 5H, arom); 7.12-7.33 (m, 5H, 'arom.). ¹³C-NMR(CDCl₃): 127.2; 128.6; 129.4; 132.7 (C arom.); 128.7; 129.2; 129.7;146.7 (′C arom.); 152.4 (3-C═S); 169.3 (5-C═O). Anal. (C₁₄H₁₀N₂S₂O) C,H, N, S.

[0121] The synthesis of compound 5 by a different synthetic route isdescribed in Slomezynska, U.; Barany, G., J. Heterocyclic. Chem., 1984,21, 241, the contents of which are incorporated herein by referencethereto.

[0122] 4-(Ethoxycarbonylmethyl)-2-methyl-1,2,4-thiadiazolidine-3,5-dione(Compound 6):

[0123] Reagents: Ethyl isothiocyanatoacetate (0.8 ml, 6.5 mmol), 35% HCl(3.1 ml), KMnO₄ (0.5 g), methyl isocyanate (0.38 ml, 6.5 mmol).

[0124] Conditions: Room temperature, 8 h.

[0125] Isolation: filtration of reaction mixture.

[0126] Purification: recrystallization from hexane.

[0127] Yield 0.28 g (20%) as white solid; mp 67-69° C. ¹H-NMR (CDCl₃):1.3 (t, 3H, CH₂CO₂CH₂CH₃, J=7.1 Hz); 3.2 (s, 3H, CH₃); (c, 2H,CH₂CO₂CH₂CH₃, J=7.1 Hz); 4.4 (s, 2H, CH₂CO₂CH₂CH₃); ¹³C-NMR (CDCl₃):14.0 (CH₂CO₂CH₂CH₃); 31.5 (CH₃); 42.7 (CH₂CO₂CH); 62.1; (CH₂CO₂CH₂CH₃);152.6 (3-C═O); 166.4 (5-C═O); 166.4 (CO₂). Anal. (C₇H₁₀N₂SO₃) C, H, N,S.

[0128] 4-(4-Methoxyphenyl)-2-methyl-1,2,4-thiadiazolidine-3,5-dione(Compound 7):

[0129] Reagents: 4-methoxyphenyl isothiocyanate (0.89 ml, 6.5 mmol), 35%HCl (3.1 ml), KMnO₄ (0.5 g), methyl isocyanate (0.38 ml, 6.5 mmol).

[0130] Conditions: Room temperature, 8 h.

[0131] Isolation: filtration of reaction mixture.

[0132] Purification: recrystallization from CH₂Cl₂ /Hexane.

[0133] Yield: 0.44 g (30%) as white solid; mp 140-144° C. ¹H-NMR(CDCl₃): 3.31 (s, 3H, CH₃); 3.80 (s, 3H, p-CH₃O-Ph); 7.02-7.32 (m, 4H,arom.). ¹³C-NMR (CDCl₃): 31.7 (CH₃); 55.5 (p-CH₃O-Ph); 114.7; 125.3;128.5; 159.9 (C arom.); 152.9 (3-C═O); 165.5 (5-C═O). Anal.(C₁₀H₁₀N₂SO₃) C, H, N, S.

[0134] 4-Benzyl-2-ethyl-1,2,4-thiadiazolidine-3,5-dione (Compound 8).

[0135] Reagents: Benzyl isothiocyanate (0.86 ml, 6.5 mmol), 35% HCl (3.1ml), KMnO₄ (0.5 g), ethyl isocyanate (0.51 ml, 6.5 mmol).

[0136] Conditions: Room temperature, 10 h. Purification: silica gelcolumn chromatography using CH₂Cl₂/Hexane (1:1) and CCTLC using CH₂Cl₂.

[0137] Yield: 0.39 g (25%) as yellow oil. ¹H-NMR (CDCl₃): 1.22 (t, 3H,CH₂′CH₃, J=7.2 Hz); 3.7 (c, 2H, ′CH₂CH₃, J=7.2 Hz); 4.8 (s, 2H, CH₂-Bn);7.32-7.44 (m, 5H, arom.) ¹³C-NMR (CDCl₃): 13.7 (CH₂′CH₃); 39.9(′CH₂CH₃); 45.8 (CH₂-Bn); 128.1; 128.6; 128.8; 135.1 (C arom.); 152.6(3-C═O); 165.9 (5-C═O). Anal. (C₁₁H₁₂N₂SO₂) C, H, N, S.

[0138] 4-Ethyl-2-isopropyl-1,2,4-thiadiazolidin-3,5-dione (Compound 9):

[0139] Synthesis of this compound is described in: Martinez, A.; Castro,A.; Cardelús, I.; Llenas, J.; Palacios, J. M., Bioorg. Med. Chem., 1997,5, 1275-1283, the contents of which are incorporated herein by referencethereto.

[0140] 4-Benzyl-2-ethyl-5-oxo-1,2,4-thiadiazolidine-3-thione (Compound10).

[0141] Reagents: Benzyl isothiocyanate (0.86 ml, 6.5 mmol), 35% HCl (3.1ml), KMnO₄ (0.5 g), ethyl isothiocyanate (0.57 ml, 6.5 mmol).

[0142] Conditions: Room temperature, 12 h.

[0143] Isolation: solvent evaporation.

[0144] Purification: silica gel column chromatography usingCH₂Cl₂/Hexane (1:2) first and preparative thin layer chromatographyusing CH₂Cl₂/Hexane (1:10) after.

[0145] Yield: 0.04 g (3%) as yellow oil. ¹H-NMR (CDCl₃): 1.2 (t, 3H,CH₂CH₃, J=7.0 Hz); 4.25 (c, 2H, CH₂CH₃, J=7.0 Hz); 4.5 (s, 2H, CH₂-Bn);7.11-7.31 (m, 5H, arom.). ¹³C-NMR (CDCl₃): 11.2 (CH₂CH₃); 46.1 (CH₂-Bn);56.2 (CH₂CH₃); 127.2; 127.3; 128.6 138.3 (C arom.); 154.3 (3-C═S); 168.7(5-C═O). Anal. (C₁₁H₁₂N₂S₂O) C, H, N, S.

[0146] 4-(4-Methylphenyl)-2-methyl-1,2,4-thiadiazolidine-3,5-dione(Compound 15).

[0147] Reagents: 4-Methylphenyl isothiocyanate (0.88 ml, 6.5 mmol), 35%HCl (3.1 ml), KMnO₄ (0.5 g), methyl isocyanate (0.38 ml, 6.5 mmol).

[0148] Conditions: Room temperature, 6 h.

[0149] Isolation: filtration of reaction mixture.

[0150] Purification: recrystallization from CH₂Cl₂ /Hexane.

[0151] Yield: 0.29 g (21%) as white solid; mp 182-184° C. ¹H-NMR(CDCl₃): 2.4 (s, 3H, p-CH₃-Ph); 3.25 (s, 3H, CH₃); 7.20-7.34 (m, 4H,arom.). ¹³C-NMR (CDCl₃): 21.1 (p-CH₃-Ph); 31.7 (CH₃); 126.7; 130.0;130.3; 139.3 (C arom.); 152.9 (3-C═O); 165.3 (5-C═O). Anal.(C₁₀H₁₀N₂SO₂) C, H, N, S.

[0152] 4-(4-Bromophenyl)-2-methyl-1,2,4-thiadiazolidine-3,5-dione(Compound 16).

[0153] Reagents: 4-Bromophenyl isothiocyanate (1.4 g, 6.5 mmol), 35% HCl(3.1 ml), KMnO₄ (0.5 g), methyl isocyanate (0.38 ml, 6.5 mmol).

[0154] Conditions: Room temperature, 9 h.

[0155] Isolation: filtration of reaction mixture.

[0156] Purification: recrystallization from hexane/CH₂Cl₂.

[0157] Yield: 0.32 g (20%) as white solid; mp 182-184° C. ¹H-NMR(CDCl₃): 3.25 (s, 3H, CH₃); 7.25-7.61 (2 d, 4H, arom., J=8.6 Hz).¹³C-NMR (CDCl₃): 31.6 (CH₃); 123.0; 128.6; 131.6; 132.5 (C arom.); 153.4(3-C═O); 165.7 (5-C═O). Anal. (C₉H₇N₂SO₂Br) C, H, N, S.

[0158] 4-(4-Fluorophenyl)-2-methyl-1,2,4-thiadiazolidine-3,5-dione(Compound 17).

[0159] Reagents: 4-Fluorophenyl isothiocyanate (1.1 g, 6.5 mmol), 35%HCl (3.1 ml), KMnO₄ (0.5 g), methyl isocyanate (0.38 ml, 6.5 mmol).

[0160] Conditions: Room temperature, 8 h.

[0161] Isolation: filtration of reaction mixture.

[0162] Purification: recrystallization from ethanol.

[0163] Yield: 0.37 g (25%) as white solid; mp 178-180° C. ¹H-NMR(CDCl₃): 3.25 (s, 3H, CH₃); 7.13-7.36 (m, 4H, arom.). ¹³C-NMR (CDCl₃):31.7 (CH₃); 116.3; 129.1; 160.9; 164.2 (C arom.); 152.5 (3-C═O); 165.2(5-C═O). Anal. (C₉H₇N₂SO₂F) C, H, N, S.

[0164] 4-(4-Chlorophenyl)-2-methyl-1,2,4-thiadiazolidine-3,5-dione(Compound 18).

[0165] Reagents: 4-Chlorophenyl isothiocyanate (1.1 g, 6.5 mmol), 35%HCl (3.1 ml), KMnO₄ (0.5 g), methyl isocyanate (0.38 ml, 6.5 mmol).

[0166] Conditions: Room temperature, 6 h.

[0167] Isolation: filtration of reaction mixture.

[0168] Purification: recrystallization from ethanol.

[0169] Yield: 0.47 g (30%) as white solid; mp 175-178° C. ¹H-NMR(CDCl₃): 3.25 (s, 3H, CH₃); 7.32-7.44 (2 d, 4H, arom., J=8.9 Hz).¹³C-NMR (CDCl₃): 31.7 (CH₃); 128.4; 129.6; 131.2; 135.1 (C arom.); 152.3(3-C═O); 165.0 (5-C═O). Anal. (C₉H₇N₂SO₂Cl) C, H, N, S.

[0170] Synthesis of 5-(2-pyridylimino)-1,2,4-thiadiazolidin-3-ones(Compounds 19-22 and 26):

[0171] A general method for the synthesis of these compounds isdescribed in Martinez, A.; Castro, A.; Cardelús, I.; Lienas, J.;Palacios, J. M., Bioorg. Med. Chem., 1997, 5, 1275-1283, the contents ofwhich are incorporated herein by reference thereto.

[0172]3-(4-Ethyl-3-oxo-2-methyl-1,2,4-thiadiazolidin-5-ylidine)aminopyridine-1-oxide(Compound 23):

[0173] A general method for the synthesis of this compound is describedin Martinez, A.; Alonso, D.; Castro, A.; Arán, J. V.; Cardelus, I.;Bãnos, J. E.; Badia, A., Arch. Pharn. Pharm. Med. Chem., 1999, 332,191-194, the contents of which are incorporated herein by referencethereto.

[0174]3-[5-(4-Ethyl-2-methyl-3-oxo)imino-1,2,4-thiadiazolidyl]-1-methyl-pyridiniumIodide (Compound 24):

[0175] A general method for the synthesis of this compound is describedin Martinez, A.; Alonso, D.; Castro, A.; Gutierrez-Puebla, E.; Baños, J.E.; Badia, A., Eur. J. Org. Chem., 2000, 675-680., the contents of whichare incorporated herein by reference thereto.

[0176]4-Ethyl-5-[imino-(1-methyl-piperidin-3-yl)]-2-methyl-1,2,4-thiadiazolidin-3-one(Compound 25):

[0177] A general method for the synthesis of this compound is describedin Martinez, A.; Alonso, D.; Castro, A.; Gutierrez-Puebla, E.; Bańos, J.E.; Badia, A., Eur. J. Org. Chem., 2000, 675-680, the contents of whichare incorporated herein by reference thereto.

[0178] Further compounds of formula (II) have been synthesised and theirGSK-3 inhibition tested. These compounds are listed in Table 3a below.TABLE 3a (II)

Com- pound IC₅₀ No. R^(a) R^(b) X Y (μM) Ref. 27 Et Et O O 25 JMC, JHC28 Et Et O S 20 JMC 29 Bn Bn O O 10 JMC, JHC 30 CH₂CO₂Et Et O O 10 below31 CH₂Ph COPh O O 3 below 32 Ph Et O NH 65 JMC 33 CH₂Ph CH₂CO₂Et O O 4below 34 4-CF₃Ph Me O O 6 JMC 35 n-Bu Et O O 70 JMC 36 CH₂Ph Et O N—OH 6below 37 3-BrPh Me O O 4 JMC 38 2-BrPh Me O O 6 JMC 39 Ph Et O NCONHEt75 JMC 40 Ph CO₂Et S NCO₂Et >10 JMC 41 CH₂CH₂Ph Et O O 8 below 42 CH₂PhH O O 50 below 43 Ph Et O O 6 ACIE 44 CH₂CO₂Et CH₂CO₂Et O O 4 below 45CH₂CO₂Et Me O O 2 below 46 CH₂CO₂Et iPr O O 7 below 47 CH₂CO₂Et Bz O O 4below 48 Naphthyl Me O O 3 JMC 49 4-NO₂—Ph Et O O 8.5 below 50 Ph Et ON—OH 100 below 51 CH₂Ph iPr O O 10 below 52 Ph Ph O O 8 ACIE 53 4-MeOPhEt O O below 54 4-MePh Et O O below 55 4-BrPh Et O O below

[0179] The synthesis of the known compounds depicted in Table 3a isdescribed in the following publications, the contents of which areincorporated herein by reference thereto:

[0180] JMC: Martinez, A; Alonso, M.; Castro, A.; Perez, C.; Moreno, F.J. J. Med. Chem. (2002) 45:1292-1299.

[0181] JCH: J. Heterocyclic Chem. (1984) 21:241.

[0182] ACIE: Angew Chem. Int. Ed. (1966) 5:672.

[0183] The synthesis of the new compounds depicted in Table 3a isdescribed below.

[0184] 4-(Ethoxycarbonylmethyl)-2-ethyl-1,2,4-thiadiazolidine-3,5-dione(Compound 30):

[0185] Reagents: Ethyl isothiocyanatoacetate (0.8 ml, 6.5 mmol), 35% HCl(3.1 ml), KMnO₄ (0.5 g), ehtyl isocyanate (0.51 ml, 6.5 mmol).

[0186] Conditions: Room temperature, 8 h.

[0187] Isolation: filtration of reaction mixture.

[0188] Purification: recrystallization from hexane.

[0189] Yield 0.52 g (34%) as white solid; mp 62-63° C. ¹H-NMR (CDCl₃):1.3 (t, 3H, CH₂CO₂CH₂CH₃, J=7.1 Hz); 1.3 (t, 3H, CH₂CH₃, J=7.1 Hz); 3.7(c, 2H, CH₂CH₃, J=7.1 Hz); 4.2 (c, 2H, CH₂CO₂CH₂CH₃, J=7.1 Hz); 4.4 (s,2H), CH₂CO₂CH₂CH₃). ¹³C-NMR (CDCl₃): 13.7 (CH₂CH₃); 14.0 (CH₂CO₂CH₂CH₃);40.1 (CH₂CH₂); 42.6 (CH₂CO₂CH₂CH₃); 62.1 (CH₂CO₂CH₂CH₃); 152.0 (3-C═O);165.7 (5-C═O); 166.4 (CO2). Anal. (C₈H₁₂N₂SO₃) C, H, N, S.

[0190] 2-Benzoyl-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (Compound 31)

[0191] Reagents: Benzyl isothiocyanate (0.86 ml, 6.5 mmol), 35% HCl (3.1ml), KMnO₄ (0.5 g), benzoyl isocyanate (0.81 ml, 6.5 mmol).

[0192] Conditions: Room temperature, 9 h.

[0193] Isolation: solvent evaporation.

[0194] Purification: silica gel column chromatography using AcOEt/Hexane(1:10).

[0195] Yield: 0.2 g (10%) as white solid. ¹H-NMR (CDCl₃): 4.8 (s, 2H,CH₂-Ph); 7.3-7.7 (m, 10H, arom.). ¹³C-NMR (CDCl₃): 45.9 (CH₂-Ph); 127.9;128.5; 128.8; 129.0; 129.2; 132.9; 134.3 (C arom); 149.0 (3-C═O); 164.7(COPh); 166.5 (5-C═O). Anal. (C₁₆H₁₂N₂SO₃) C, H, N, S.

[0196] 4-Benzyl-2-(Ethoxycarbonylmethyl)-1,2,4-thiadiazolidine-3,5-dione(Compound 33):

[0197] Reagents: Benzyl isothiocyanate (0.86 ml, 6.5 mmol), 35% HCl (3.1ml), KMnO₄ (0.5 g), Ethyl isocyanatoacetate (0.73 ml, 6.5 mmol).

[0198] Conditions: Room temperature, 9 h.

[0199] Isolation: solvent evaporation.

[0200] Purification: silica gel column chromatography using AcOEt/Hexane(1:6).

[0201] Yield: 0.75 g (39%) as colorless oil. ¹H-NMR (CDCl₃): 1.25 (t,3H, CH₂CO₂CH₂CH₃, J=7.1 Hz); 4.21 (c, 2H, CH₂CO₂CH₂CH₃, J=7.1 Hz); 4.30(s, 2H, CH₂CO₂CH₂CH₃); 4.8 (s, CH₂-Ph); 7.3-7.5 (m, 5H, arom.). ¹³C-NMR(CDCl₃): 13.7 (CH₂CO₂CH₂CH₃); 45.3 (CH₂CO₂CH₂CH₃); 45.7 (CH₂-Ph); 127.3;128.3; 128.4; 134.7 (C arom.); 153.3 (3-C═O); 165.7 (5-C═O); 166.8(CH₂CO₂CH₂CH₃). Anal. (C₁₃H₁₄N₂SO₄) C, H, N, S.

[0202] 4-Benzyl-2-ethyl-1,2,4-thiadiazolidine-3-one-5-oxime (Compound36):

[0203] Reagents: 5-chloro-4-benzyl-2-ethyl-3-oxo-1,2,4-thiadiazoliumchloride (1.24 g, 4.5 mmol), hydroxylamine hydrochloride (0.35 g, 5mmol), pyridine (0.8 ml, 10 mmol).

[0204] Conditions: Room temperature, 12 h.

[0205] Isolation: solvent evaporation.

[0206] Purification: silica gel column chromatography using AcOEt/Hexane(1:6).

[0207] Yield: 0.10 g (9%) as yellow oil. ¹H-NMR (CDCl₃): 1.22 (t, 3H,CH₂CH₃, J=7.1 Hz); 3.60 (c, 2H, CH₂CH₃, J=7.1 Hz); 4.78 (s, 2H, CH₂Ph);6.57 (s, 1H, N-OH); 7.24-7.40 (m, 5H, arom). ¹³C-NMR (CDCl₃): 13.5(CH₂CH₃); 40.2 (CH₂CH₃); 46.9 (CH₂Ph); 127.8; 128.4; 128.5; 135.2 (Carom.); 152.2 (3-C═O); 154.6 (5-C═NOH). Anal. (C₁₁H₁₃N₃SO₂) C, H, N, S.

[0208] 2-Ethyl-4-phenethyl-1,2,4-thiadiazolidine-3,5-dione (Compound41).

[0209] Reagents: phenethyl isothiocyanate (0.97 ml, 6.5 mmol), 35% HCl(3.1 ml), KMnO₄ (0.5 g), ethyl isocyanate (0.51 ml, 6.5 mmol).

[0210] Conditions: Room temperature, 10 h.

[0211] Isolation: solvent evaporation.

[0212] Purification: silica gel column chromatography using AcOEt/Hexane(1:6).

[0213] Yield: 0.26 g (16%) as yellow oil. ¹H-NMR (CDCl₃): 1.22 (t, 3H,CH₂CH₃, J=7.1 Hz); 2.95 (m, 2H, CH₂CH₂Ph); 3.63 (c, 2H, CH₂CH₃, J=7.1Hz); 3.89 (m, 2H, CH₂CH₂Ph); 7.20-7.29 (m, 5H, arom). ¹³C-NMR (CDCl₃):13.6 (CH₂CH₃); 33.6 (CH₂CH₂Ph); 39.9 (CH₂CH₂Ph); 43.5 (CH₂CH₃); 126.6;128.5; 128.8; 137.3 (C arom.); 152.7 (3-C═O); 165.7 (5-C═O) Anal.(C₁₂H₁₄N₂SO₂) C, H, N, S.

[0214] 4-Benzyl-1,2,4-thiadiazolidine-3,5-dione (Compound 42).

[0215] Reagents: Benzyl isothiocyanate (0.81 ml, 6.5 mmol), 35% HCl (3.1ml), KMnO₄ (0.5 g), ethyl isocyanatoformate (0.69 ml, 6.5 mmol).

[0216] Conditions: Room temperature, 8 h.

[0217] Isolation: Deprotection in situ of the nitrogen with acidconditions.

[0218] Purification: preparative centrifugal circular thin layerchromatography (CCTLC) using CH₂Cl₂.

[0219] Yield: 0.01 g (1%) as colourless oil. ¹H-NMR (CDCl₃): 4.4 (s, 2H,CH₂Ph); 6.1 (br, NH); 7.1-7.3 (m, 5H, arom.). ¹³C-NMR (CDCl₃): 51.1(CH₂Ph); 127.1; 128.3; 129.2; 139.6 (C arom.); 152.1 (3-C═O); 165.6(5-C═O). Anal. (C₉H₈N₂SO₂) C, H, N, S.

[0220]4-(Ethoxycarbonylmethyl)-2-(ethoxycarbonylmethyl)-1,2,4-thiadiazolidine-3,5-dione(Compound 44).

[0221] Reagents: Ethyl isothiocyanatoacetate (0.8 ml, 6.5 mmol), 35% HCl(3.1 ml), KMnO₄ (0.5 g), ethyl isocyanatoacetate (0.73 ml, 6.5 mmol).

[0222] Conditions: Room temperature, 9 h.

[0223] Isolation: solvent evaporation.

[0224] Purification: silica gel column chromatography using AcOEt/Hexane(1:3).

[0225] Yield: 0.90 g (48%) as white solid; mp. 72-74° C. ¹H-NMR (CDCl₃):1.25 (t, 3H, ′CH₂CO₂CH₂CH₃, J=7.1 Hz); 1.26 (t, 3H, CH₂CO₂CH₂CH₃, J=7.1Hz); 4.18 (c, 2H, ′CH₂CO₂CH₂CH₃, J=7.1Hz); 4.20 (c, 2H, CH₂CO₂CH₂CH₃,J=7.1); 4.3 (s, 2H, ′CH₂CO₂CH₂CH₃); 4.4 (s, 2H, CH₂CO₂CH₂CH₃). ¹³C-NMR(CDCl₃): 14.0 (′CH₂CO₂CH₂CH₃); 14.0 (CH₂CO₂CH₂CH₃); 42.7 (CH₂CO₂CH₂CH₃);45.6 (′CH₂CO₂CH₂CH₃); 62.1 (′CH₂CO₂CH₂CH₃); 62.1 (CH₂CO₂CH₂CH₃); 153.0(3-C═O); 165.7 (5-C═O); 166.1 (CH₂CO₂CH₂CH₃); 166.8 (′CH₂CO₂CH₂CH₃).Anal. (C₁₀H₁₄N₂SO₆) C, H, N, S.

[0226] 4-(Ethoxycarbonylmethyl)-2-methyl-1,2,4-thiadiazolidine-3,5-dione(Compound 45).

[0227] Reagents: Ethyl isothiocyanatoacetate (0.8 ml, 6.5 mmol), 35% HCl(3.1 ml), KMnO₄ (0.5 g), methyl isocyanate (0.38 ml, 6.5 mmol).

[0228] Conditions: Room temperature, 8 h.

[0229] Isolation: filtration of reaction mixture.

[0230] Purification: recrystallization from hexane.

[0231] Yield 0.28 g (20%) as white solid; mp 67-69° C. ¹H-NMR (CDCl₃):1.3 (t, 3H, CH₂CO₂CH₂CH₃, J=7.1 Hz); 3.2 (s, 3H, CH₃); 4.2 (c. 2H,CH₂CO₂CH₂CH₃, J=7.1 Hz); 4.4 (s, 2H, CH₂CO₂CH₂CH₃). ¹³C-NMR (CDCl₃):14.0 (CH₂CO₂CH₂CH₃); 31.5 (CH₃); 42.7 (CH₂CO₂CH₂CH₃); 62.1(CH₂CO₂CH₂CH₃); 152.6 (3-C═O); 166.4 (5-C═O); 166.4 (CO₂). Anal.(C₇H₁₀N₂SO₃) C, H, N, S.

[0232]4-(Ethoxycarbonylmethyl)-2-isopropyl-1,2,4-thiadiazolidine-3,5-dione(Compound 46).

[0233] Reagents: Ethyl isothiocyanatoacetate (0.8 ml, 6.5 mmol), 35% HCl(3.1 ml), KMnO₄ (0.5 g), isopropyl isocyanate (0.64 ml, 6.5 mmol).

[0234] Conditions: Room temperature, 9 h.

[0235] Isolation: filtration of reaction mixture.

[0236] Purification: recrystallization from hexane.

[0237] Yield: 0.48 g (30%) as white solid; mp 80-82° C. ¹H-NMR (CDCl₃):1.3 (t, 3H, CH₂CO₂CH₂CH₃, J=7.1 Hz); 1.3 (d, 6H, CH(CH₃)₂, J=7.1 Hz);3.8 (sp, 1H, CH(CH₃)₂, J=7.1 Hz); 4.1 (c, 2H, CH₂CO₂CH₂CH₃, J=7.1 Hz);4.4 (s, 2H, CH₂CO₂CH₂CH₃). 13C-NMR (CDCl₃): 13.6 (CH₂CO₂CH₂CH₃);20.1(CHCH₃CH₃); 45.1 (CHCH₃CH₃); 48.2 (CH₂CO₂CH₂CH₃); 59.2(CH₂CO₂CH₂CH₃); 153.0 (3-C═O); 165.6 (5-C═O); 167.3 (CO₂). Anal.(C₉H₁₄N₂SO₄) C, H, N, S.

[0238]2-Benzoyl-4-(ethoxycarbonylmethyl)-1,2,4-thiadiazolidine-3,5-dione(Compound 47).

[0239] Reagents: Ethyl isothiocyanatoacetate (0.8 ml, 6.5 mmol), 35% HCl(3.1 ml), KMnO₄ (0.5 g), benzoyl isocyanate (0.81 ml, 6.5 mmol).

[0240] Conditions: Room temperature, 9 h.

[0241] Isolation: solvent evaporation.

[0242] Purification: silica gel column chromatography using AcOEt/Hexane(1:5).

[0243] Yield: 0.07 g (4%) as colorless oil. ¹H-NMR (CDCl₃): 1.26 (t, 3H,CH₂CO₂CH₂CH₃, J=7.1 Hz); 4.2 (c, 2H, CH₂CO₂CH₃CH₃, J=7.1 Hz); 4.4 (s,2H, CH₂CO₂CH₂CH₃); 7.4-7.7 (m, 5H, arom). ¹³C-NMR (CDCl₃): 13.9(CH₂CO₂CH₂CH₃); 42.3 (CH₂CO₂CH₂CH₃); 62.4 (CH₂CO₂CH₂CH₃); 127.9; 129.2;131.7; 133.1 (C arom); 148.6 (3-C═O); 164.4 (5-C═); 166.4(CH₂CO₂CH₂CH₃); 165.7(CO-Ph). Anal. (C₁₃H₁₂N₂SOs) C, H, N, S.

[0244] 2-Ethyl-4-(4-nitrophenyl)-1,2,4-thiadiazolidine-3,5-dione(Compound 49).

[0245] Reagents: 4-nitrophenyl isothiocyanate (1.17 g, 6.5 mmol), 35%HCl (3.1 ml), KMnO₄ (0.5 g), ethyl isocyanate (0.51 ml, 6.5 mmol) inTHF.

[0246] Conditions: Room temperature, 10 h.

[0247] Isolation: solvent evaporation.

[0248] Purification: silica gel column chromatography using AcOEt/Hexane(1:4).

[0249] Yield: 0.26 g (11%) as yellow solid; mp. 117-118° C. ¹H-NMR(CDCl₃): 1.34 (t, 3H, CH₂CH₃, J=7.1 Hz); 3.77 (c, 2H, CH₂CH₃, J=7.1 Hz);7.6-8.4 (m, 5H, arom). ¹³C-NMR (CDCl₃): 13.6 (CH₂CH₃; 40.3 (CH₂CH₃);124.3; 127.6; 137.9; 147.1 (C arom.); 150.9 (3-C═O); 164.8 (5-C═O).Anal. (C₁₀H₉N₃SO₄) C, H, N, S.

[0250] 2-Ethyl-4-phenyl-1,2,4-thiadiazolidine-3-one-5-oxime (Compound50).

[0251] Reagents: 5-chloro-4-phenyl-2-ethyl-3-oxo-1,2,4-thiadiazoliumchloride (1.24 g, 4.5 mmol), hydroxylamine hydrochloride (0.35 g, 5mmol), pyridine (0.8 ml, 10 mmol).

[0252] Conditions: Room temperature, 12 h.

[0253] Isolation: solvent evaporation.

[0254] Purification: silica gel column chromatography using AcOEt/Hexane(1:4) first, and then preparative centrifugal circular thin layerchromatography (CCTLC) using AcOEt/Hexane (1:3).

[0255] Yield: 0.13 g (12%) as yellow solid; mp. 115-117° C. ¹H-NMR(CDCl₃): 1.28 (t, 3H, CH₂CH₃, J=7.1 Hz); 3.64 (c, 2H, CH₂CH₃, J=7.1 Hz);6.65 (s, 1H, N-OH); 7.24-7.50 (m, 5H, arom). ¹³C-NMR (CDCl₃): 13.4(CH₂CH₃); 40.2 (CH₂CH₃); 127.0; 128.6; 129.2; 133.8 (C arom.); 152.4(3-C═O); 153.5 (5-C═NOH). Anal. (C₁₀H₁₁N₃SO₂) C, H, N, S.

[0256] 4-Benzyl-2-isopropyl-1,2,4-thiadiazolidine-3,5-dione (Compound51).

[0257] Reagents: Benzyl isothiocyanate (0.81 ml, 6.5 mmol), 35% HCl (3.1ml), KMnO₄ (0.5 g), isopropyl isocyanate (0.64 ml, 6.5 mmol).

[0258] Conditions: Room temperature, 8 h.

[0259] Isolation: solvent evaporation.

[0260] Purification: silica gel column chromatography using AcOEt/Hexane(1:3).

[0261] Yield: 0.50 g (31%) as yellow oil. ¹H-NMR (CDCl₃): 1.2 (d, 6H,CH(CH₃)₂, J=6.6 Hz); 4.7 (sp, 1H, CH(CH₃)₂, J=6.6 Hz); 4.8 (s, 2H,CH₂Ph); 7.2-7.4 (m, 5H, arom.). ¹³C-NMR (CDCl₃): 21.2 ((CH₃)₂CH); 45.5(CH(CH₃)₂); 47.0 (CH₂Ph); 127.8; 128.4; 128.5; 135.0 (C arom.); 151.9(3-C═O); 165.8 (5-C═O). Anal. (C₁₂H₁₄N₂SO₂) C, H, N, S.

[0262] 2-Ethyl-4-(4-methoxyphenyl)-1,2,4-thiadiazolidine-3,5-dione(Compound 53).

[0263] Reagents: 4-Methoxyphenyl isothiocyanate (0.89 ml, 6.5 mmol), 35%HCl (3.1 ml), KMnO₄ (0.5 g), ethyl isocyanate (0.51 ml, 6.5 mmol).

[0264] Conditions: Room temperature, 8 h.

[0265] Isolation: solvent evaporation.

[0266] Purification: silica gel column chromatography using AcOEt/Hexane(1:4).

[0267] Yield: 0.344 g (21%) as white solid. ¹H-NMR (CDCl₃): 1.2 (t, 3H,CH₃CH₂ J=7.2 Hz); 3.6 (c, 2H, CH₃CH₂, J=7.2 Hz); 3.7 (s, 3H, p-CH₃O-Ph);6.9-7.2 (2d, 4H, arom., J=9.4 Hz). ¹³C-NMR (CDCl₃): 14.2 (CH₃CH₂); 40.6(CH₃CH₂); 55.8 (p-CH₃O-Ph); 114.7; 125.6; 128.7; 159.9 (C arom.); 152.4(3-C═O); 165.8 (5-C═O). Anal. (C₁₁H₁₂N₂SO₃) C, H, N, S.

[0268] 2-Ethyl-4-(4-methylphenyl)-1,2,4-thiadiazolidine-3,5-dione(Compound 54).

[0269] Reagents: 4-Methylphenyl isothiocyanate (0.88 ml, 6.5 mmol), 35%HCl (3.1 ml), KMnO₄ (0.5 g), ethyl isocyanate (0.51 ml, 6.5 mmol).

[0270] Conditions: Room temperature, 8 h.

[0271] Isolation: solvent evaporation.

[0272] Purification: silica gel column chromatography using AcOEt/Hexane(1:4).

[0273] Yield: 0.37 g (25%) as white solid. ¹H-NMR (CDCl₃): 1.3 (t, 3H,CH₃CH₂, J=7.3 Hz); 2.4 (s, 3H, p-CH₃-Ph); 3.7 (c, 2H, CH₃CH₂, J=7.3 Hz);7.20-7.34 (m, 4H, arom.). ¹³C-NMR (CDCl₃): 13.9 (CH₃CH₂); 21.3(p-CH₃-Ph); 40.3 (CH₃CH₂); 126.8; 129.8; 129.9; 139.1 (C arom.); 152.0(3-C═O); 165.4 (5-C═O). Anal. (C₁₁H₁₂N₂SO₂) C, H, N, S.

[0274] (4-Bromophenyl)-2-ethyl-1,2,4-thiadiazolidine-3,5-dione (Compound55).

[0275] Reagents: 4-Bromophenyl isothiocyanate (1.4 g, 6.5 mmol), 35% HCl(3.1 ml), KMnO₄ (0.5 g), ethyl isocyanate (0.51 ml, 6.5 mmol).

[0276] Conditions: Room temperature, 9 h.

[0277] Isolation: solvent evaporation.

[0278] Purification: silica gel column chromatography using AcOEt/Hexane(1:4).

[0279] Yield: 0.256 g (13%) as white solid. ¹H-NMR (CDCl₃): 1.3 (t, 3H,CH₃CH₂, J=7.2 Hz); 3.7 (c, 2H, CH₃CH₂, J=7.2 Hz); 7.3-7.6 (2 d, 4H,arom., J=8.8 Hz). ¹³C-NMR (CDCl₃): 13.8 (CH₃CH₂); 40.4 (CH₃CH₂); 122.9;128.5; 131.5; 132.3 (C arom.); 151.4 (3-C═O); 164.9 (5-C═O). Anal.(C₁₀H₉N₂SO₂Br) C, H, N, S.

[0280] GSK-3 inhibition: The experiments of inhibition were alsoperformed at variable concentrations of ATP (up to 50 μM) and in allcases the same value of IC₅₀ were obtained. Thus could suggest thatthiadiazolinediones do not compete with ATP in the binding to GSK-3.

[0281] The first four compounds were assayed for inhibition of otherenzymes.

[0282] Protein kinase A (PKA) inhibition: The potential inhibition ofthis enzyme is evaluated by determining the esthatmine phosphorylationby the protein kinase A (PKA). The esthatmine was purified following theprocedure described by Belmont and Mitchinson (Belmont, L. D.;Mitchinson, T. J. “Identification of a protein that interact withtubulin dimers and increases the catastrophe rate of microtubule”, Cell,1996, 84, 623-631).

[0283] Concretely, it was used purified PKA (Sigma, catalytic subunitfrom bovine heart (p 2645)) and 10-15 μg of substrate (esthatmine) in a25 μl total volume of buffer solution containing 20 μM (γ-³²P)ATP. ThecAMP kinase protein (100 ng/reaction) was performed in 50 μl of 25 mMhepes, pH 7.4, 20 mM MgCl₂, 2 mM EGTA, 2 mM dithiothreitol, 0.5 mMNa₃VO₄. After the reaction took place, a quenching buffer was added, thereaction mixture was boiled at 100° C. during 5 minutes and thephosphorylated protein was characterized by gel electrophoresys andquantified by autoradiographia.

[0284] In these conditions none of the compounds assayed showed anyinhibition of PKA.

[0285] Protein kinase C (PKC) inhibition: The potential inhibition ofthis enzyme is evaluated by determining the phosphorylation of thepeptide PANKTPPKSPGEPAK (Woodgett, J. R. “Use of peptides for affinitypurification of protein-serine kinases”, Anal. Biochem., 1989, 180,237-241) by the protein kinase C (PKC) using phosphatidyl serine asstimulating agent. The method followed is the same described above forGSK-3.

[0286] Concretely, it was used PKC purified from rat brains followingthe method described by Walsh (Walsh, M. P.; Valentine, K. A.; Nagi, P.K.; Corruthers, C. A.; Hollenberg, M. D. Biochem. J., 1984, 224,117-127) and 1-10 mM of substrate in a total volume of 25 μl ofadecuated buffer solution containing 10 μM (γ-³²P)ATP.

[0287] In these conditions none of the compounds assayed showed anyinhibition of PKC.

[0288] Casein kinase 2 (CK-2) inhibition: The phosphorylating activityof this enzyme against esthatmine has been measured using CK-2 purifiedfrom bovine brains, following the method described by Alcazar (Alcazar,A.; Marín, E.; Lopez-Fando, J.; Salina, M. “An improved purificationprocedure and properties of casein kinase II from brain”, Neurochem.Res., 1988, 13, 829-836), with 3.6 AM of substrate in a total volume of25 μl of an adequate buffer solution containing 20 μM (γ-³²P)ATP. TheCK-2 assays were performed with esthatmine as substrate (see PKAdetermination) in 50 μl of 25 mM Hepes, pH 7.4, 20 mM MgCl₂, 2 mM EGTA,2 mM dithiothreitol, 0.5 mM Na₃VO₄, and 100 ng of purified CK-2. Afterthe reaction took place, it was followed the same method described forPKA.

[0289] In these conditions none of the compounds assayed showed anyinhibition of CK-2.

[0290] Cyclin dependent protein kinase 2 (Cdc2) inhibition: Thephosphorylating activity of this enzyme against histone H1 has beenmeasured using Cdc2 (Calbiochem) following the method described byKobayashi (Kobayashi, H.; Stewart, E.; Poon, R. Y.; Hunt, T. “Cyclin Aand cyclin B dissociate from p34cdc2 with half-times of 4 and 15 h,respectively, regardless of the phase of the cell cycle”, J. Biol.Chem., 1994, 269, 29153-29160), with 1 μg/μl of substrate in a totalvolume of 25 μl of the adequate buffer solution containing 20 μM(γ-³²P)ATP. The Cdc2 assays were performed with histone H1 as substrate(see PKA determination) in 50 μl of buffer pH 7.5, 50 mM Tris-HCl, 10 mMCl₂Mg, 1 mM DTT, 1 mM EGTA, 100 μM ATP, 0.01% BRIJ-35. After thereaction took place, it was followed the same method described for PKA.

[0291] In these conditions none of the compounds assayed showed anyinhibition of Cdc2.

Example 2 Analysis of the Neurites Growth After the Drug Treatment

[0292] Cells were maintained in a Dulbecco medium (DEMEM) with a 10%fethal bovine serum, glutamine (2 mM) and antibiotics. For the analysisof the potential GSK-3 inhibition in vivo, mice neuroblastoms N₂Acultures (Garcia-Perez, J.; Avila, J.; Diaz-Nido, J. “Lithium inducesmorphological differentiation of mouse neuroblastoma”, J. Neurol. Res.,1999, 57, 261-270) were used. The test compounds were added to thesecells cultures. This cell line has the particularity of expressed acertain kind of neuronal phenotype (neuritic extensions) after theaddition of lithium chloride (10 mM), a known GSK-3 inhibitor. After 2-3days of culture, it was check the effect of the tested compoundsgathered in table I. It was observed that the generation of neuriticextension in the same extension than when lithium was added. That factconfirms the in vivo GSK-3 inhibition of the compounds of the invention.

Example 3 Cell Cycle Blockade

[0293] In parallel, the potential interference of these compounds withthe cell cycle was studied on N₂A cells. The cell culture was maintainedin a Dulbecco medium (DEMEM) with a 10% fethal bovine serum, glutamine(2 mM) and antibiotics.

[0294] The first four compounds of general formula (I) gathered in Table3 were assayed in the described conditions and shown ability to inhibitthe cell cycle at an inhibitor concentration comprised between 100 nMand 1 μM. The cellular blockade was initially observed at concentrationscomprised between 100-200 nM and was totally effective at 1 μM.

[0295] The tested compounds was non toxic in stationary fibroblastculture MRC-5 after 10 days of continue exposure to the inhibitors.

Example 4 GSK-3 Inhibition of Further Compounds GSK-3 Inhibition Data

[0296] TABLE 4 Family Compound IC₅₀ (μM) A

1-A >100 B

1-B 12 C

1-C >100 D

R = H (1-D) R = CH₂Ph (2-D) R = Me (3-D) 6 1 5 E

R = H; X, Y = O (1-E) R = CH₂Ph; X, Y = O (2-E) R = CH₂Ph; X = O; Y = H(3-E) >100 >100 >100 F

R = H (1-F) R = CH₂Ph (2-F) >100 >100 G

R = (1-G) R = Me (2-G) R = CH₂CO₂H (3-G) R = CH₂Ph (4-G) R = CH₂CH₂Ph(5-G) R = CH₂COPh (6-G) >100 >100 >100 25 35 50 H

R = H (1-H) R = Me (2-H) >100 >100

Detailed Synthesis of Some of the Compounds Depicted in Table 4(Families A-H)

[0297] Synthesis of the Compounds of Family D:

[0298] N-Benzylmaleimide (compound 2-D): described in Walker, M. A.,Tetrahedron Lett., 1994, 35, 665-668.

[0299] Synthesis of the Compounds of Family G:

[0300] (4-oxo-2-thioxo-thiazolidin-3-yl)-acetic acid (compound 3-G):Girard, M. L.; Dreux, C., Bull. Soc. Chim. Fr, 1968, 3461-3468.

[0301] 3-Benzyl-2-thioxo-thiazolidin-4-one (Compound 4-G):

[0302] Reagents: Rhodanine (53 mg, 0.4 mmol), triethylamine (0.05 ml)and benzyl bromide (68

[0303] mg, 0.4 mmol) in 25 ml of acetone.

[0304] Conditions: Refluxed for 6 h.

[0305] Isolation: Add water and extract with ethyl acetate (3×5 ml).

[0306] Purification: preparative centrifugal circular thin layerchromatography (CCTLC) using CH₂Cl₂/Hexane (2:1).

[0307] Yield: 10 mg (10%) as yellow oil. ¹H-NMR (CDCl₃): 3.9 (s, 2H);5.2 (s, 2H, CH₂Ph); 7.3-7.4 (m, 5H, arom). ¹³C-NMR (CDCl₃): 35.4 (CH₂);47.6 (CH₂Ph); 128.2; 128.6; 129.1; 134.7 (C arom); 153.8 (C═O); 173.8(C═S). Anal. (C₁₀H₉NS₂O) C, H, N, S.

[0308] An alternative method for the synthesis of this compound isdescribed in J. Parkt. Chem., 1910, 81, 456, the contents of which areincorporated herein by reference thereto.

[0309] 3-Phenethyl-2-thioxo-thiazolidin-4-one (Compound 5-G):

[0310] Reagents: Rhodanine (133 mg, 1 mmol), triethylamine (0.14 ml) andphenethyl bromide (0.14 ml, 1 mmol) in 25 ml of acetone.

[0311] Conditions: Refluxed for 12 h.

[0312] Isolated: Add water and extract with ethyl acetate (3×10 ml).

[0313] Purification: preparative centrifugal circular thin layerchromatography (CCTLC) using CH₂Cl₂/Hexane (2:1).

[0314] Yield: 10 mg (4%) as yellow oil. ¹H-NMR (CDCl₃): 2.9 (t, 2H,CH₂CH₂Ph, J=8.1), 3.9 (s, 2H); 4.2 (s, 2H, CH₂CH₂Ph, J=8.1); 7.4-7.9 (m,5H, arom). ¹³C-NMR (CDCl₃): 32.6 (CH₂CH₂Ph); 35.3 (CH₂); 45.7(CH₂CH₂Ph); 126.8; 128.6; 128.6; 137.4 (C arom); 173.5 (C═O); 200.9(C═S). Anal. (C₁₁H₁₁NS₂O) C, H, N, S.

[0315] An alternative method for the synthesis of this compound isdescribed in: Buck, Leonard, J. Am. Chem. Soc., 1931, 53, 2688-2690, thecontents of which are incorporated herein by reference thereto.

[0316] 3-Phenacyl-2-thioxo-thiazolidin-4-one (Compound 6-G):

[0317] Reagents: Rhodanine (133 mg, 1 mmol), K₂CO₃ (excess) andacetophenone bromide (199 mg, 1 mmol) in 25 ml of acetone.

[0318] Conditions: Stirred at room temperature for 3 h.

[0319] Isolation: Filtration of the carbonate and evaporation of thesolvent to dryness in vacuo.

[0320] Purification: preparative centrifugal circular thin layerchromatography (CCTLC) using CH₂Cl₂.

[0321] Yield: 38 mg (15%) as brown oil. ¹H-NMR (CDCl₃): 3.9 (s, 2H); 4.2(s, 2H, CH₂COPh); 7.4-7.9 (m, 5H, arom). ¹³C-NMR (CDCl₃): 37.6 (CH₂);45.3 (CH₂COPh); 128.6; 128,7; 133.5; 135.3 (C arom); 170.5 (C═O); 194.1(CH₂COPh); 197.6 (C═S). Anal. (C₁₁H₉NS₂O₂) C, H, N, S.

[0322] Further compounds of formula (III) have been synthesised andtheir GSK-3 inhibition tested. These compounds are listed in Tables 4aand 4b below. TABLE 4a This lists further compounds of Family D in Table4 above, ie those compounds of formula:

wherein R is as listed in the Table. Compound No. R GSK-3β IC₅₀ (μM) 4-D(CH₂)₂Ph 2 5-D (CH₂)₃Ph 3 6-D (CH₂)₅Ph 3 7-D p-OCH₃-Bn 2.5 8-Dp-OCH₃—(CH₂)₂Ph 3 9-D CH₂CO₂Et 3

General Method for the Synthesis of N-alkyl-maleimides

[0323] This method is described in: Walker, M. A., Tetrahedron Lett.,1995, 35, 665-668, the contents of which are incorporated herein byreference thereto.

[0324] A 50 ml round bottom flask was charged with Ph₃P to which wasadded 25 ml of dry THF. The resulting clear solution was cooled to −70°C. under a nitrogen atmosphere. DIAD or DEAD, depending on the case, wasadded over 2-3 min. The yellow reaction mixture was stirred 5 min afterwhich the corresponding alkyl alcohol was added over 1 min and stirredfor 5 min. Maleimide was then added to the reaction mixture as solid.The resulting suspension was allowed to remain at −70° C. for 5 min,during which time most of the maleimide dissolved. The cooling bath wasthen removed, and the reaction was stirred overnight at ambienttemperature. The solvent was evaporated to dryness in vacuo and theresidue purified by silica gel column chromatography using as eluantmixtures of solvents in the proportions indicated for each particularcase.

[0325] N-phenethylmaleimide (compound 4-D): Walker, M. A., TetrahedronLett., 1995, 35, 665-668.

[0326] N-(3-phenylpropyl)maleimide (Compound 5-D):

[0327] Reagents: Ph₃P (0.65 g, 2.5 mmol), DIAD (0.5 ml, 2.5 mmol),3-phenyl-1-propanol (0.48 ml, 3.75 mmol) and maleimide (0.24 g, 2.5mmol).

[0328] Conditions: Room temperature, overnight.

[0329] Purification: silica gel column chromatography using AcOEt/Hexane(1:4).

[0330] Yield: 0.20 g (37%) as white solid; mp 79-80° C. ¹H-NMR (CDCl₃):1.92 (q, 2H, CH₂CH₂CH₂Ph, J=7.1 Hz); 2.60 (t, 2H, CH₂CH₂CH₂Ph, J=7.1Hz); 3.55 (t, 2H, CH₂CH₂CH₂Ph, J=7.1 Hz); 6.27 (d, 2H, CH═CH, J=6.4 Hz);7.12-7.28 (m, 5H, arom). ¹³C-NMR (CDCl₃): 29.6 (CH₂—CH₂CH₂Ph); 32.8(CH₂CH₂CH₂Ph); 37.4 (CH₂CH₂CH₂Ph); 125.8; 128.1; 128.2; 140.7 (C arom);133.7 (C═C); 170.6 (C═O) Anal. (C₁₃H₁₃NO₂) C, H, N, S.

[0331] N-(5-phenylpentyl)maleimide (Compound 6-D):

[0332] Reagents: Ph₃P (0.65 g, 2.5 mmol), DIAD (0.5 ml, 2.5 mmol),5-phenyl-1-pentanol (0.63 ml, 3.75 mmol) and maleimide (0.24 g, 2.5mmol).

[0333] Conditions: Room temperature, overnight.

[0334] Purification: silica gel column chromatography using AcOEt/Hexane(1:4).

[0335] Yield: 0.32 g (52%) as white-yellow solid; mp 49-51° C. ¹H-NMR(CDCl₃): 1.20-138 (m, 2H, CH₂CH₂CH₂CH₂—CH₂Ph); 1.52-2.02 (m, 4H,CH₂CH₂CH₂CH₂CH₂Ph); 2.57 (t, 2H, CH₂CH₂CH₂CH₂—CH₂Ph, J=7.3 (t, 2H,CH₂CH₂CH₂CH₂CH₂Ph, J=7.3 Hz); 6.65 (d, 2H, CH═CH, J=6.4 Hz); 7.11-7.28(m, 5H, arom). ¹³C-NMR (CDCl₃): 25.9 (CH₂CH₂CH₂—CH₂CH₂Ph); 28.0(CH₂CH₂CH₂—CH₂CH₂Ph); 30.6 (CH₂CH₂CH₂CH₂—CH₂Ph); 35.4(CH₂CH₂CH₂CH₂—CH₂Ph);37.3 (CH₂CH₂CH₂CH₂CH₂Ph); 125.4; 127.9; 128.0;142.0 (C arom); 133.6 (C═C); 170.5 (C═O) Anal. (C₁₅H₁₇NO₂) C, H, N, S.

[0336] N-(p-methoxybenzyl)maleimide (Compound 7-D):

[0337] Reagents: Ph₃P (1.31 g, 5 mmol), DEAD (0.8 ml, 5 mmol),p-methoxybenzyl alcohol (0.93 ml, 7.5 mmol) and maleimide (0.48 g, 5mmol).

[0338] Conditions: Room temperature, overnight.

[0339] Purification: silica gel column chromatography using AcOEt/Hexane(1:3).

[0340] Yield: 0.50 g (46%) as white solid; mp. 99-102° C. ¹H-NMR(CDCl₃): 3.74 (s, 3H, OCH₃); 4.58 (s, 2H, CH₂Ph); 6.65 (d, 2H, CH═CH,J=6.4 Hz); 6.8-7.2 (m, 4H, arom). ¹³C-NMR (CDCl₃): 40.4 (CH₂Ph-OCH₃);54.8 (—OCH₃); 113.6; 128.2; 129.5; 158.8 (C arom); 133.8 (C═C); 170.1(C═O) Anal. (C₁₂H₁₁NO₃) C, H, N, S.

[0341] N-(p-Methoxyphenethyl)maleimide (Compound 8-D)

[0342] Reagents: Ph₃P (1.31 g, 5 mmol), DEAD (0.8 ml, 5 mmol),p-methoxyphenethyl alcohol (1.2 g, 7.5 mmol) and maleimide (0.48 g, 5mmol).

[0343] Conditions: Room temperature, overnight.

[0344] Purification: silica gel column chromatography using AcOEt/Hexane(1:5).

[0345] Yield: 0.71 g (60%) as yellow solid; mp. 79-81° C. ¹H-NMR(CDCl₃): 2.80 (m, 2H, CH₂CH₂Ph); 3.70 (m, 2H, CH₂CH₂Ph); 3.75 (s, 3H,OCH₃); 6.63 (d, 2H, CH═CH, J=6.4 Hz); 6.8-7.1 (m, 4H, arom). ¹³C-NMR(CDCl₃): 33.5 (CH₂CH₂Ph-OCH₃); 39.2 (CH₂CH₂Ph—OCH₃); 55.5 (—OCH₃);114.0; 129.7; 129.9; 158.4 (C arom); 133.9 (C═C); 170.5 (C═O). Anal.(C₁₃H₁₃NO₃) C, H, N, S.

[0346] N-(Ethoxycarbonylmethyl)maleimide (Compound 9-D)

[0347] Reagents: Ph₃P (1.31 g, 5 mmol), DEAD (0.8 ml, 5 mmol), ethylglycollate (0.71 ml, 7.5 mmol) and maleimide (0.48 g, 5 mmol).

[0348] Conditions: Room temperature, overnight.

[0349] Purification: silica gel column chromatography using AcOEt/Hexane(1:3).

[0350] Yield: 0.30 g (33%) as colourless oil. ¹H-NMR (CDCl₃): 1.25 (t,3H, CH₂CO₂CH₂CH₃, J=7.1 Hz); 4.20 (c, 2H, CH₂CO₂CH₂CH₃, J=7.1 Hz); 4.24(s, 2H,. CH₂CO₂CH₂CH₃); 6.76 (d, 2H, CH═CH, J=6.4 Hz). ¹³C-NMR (CDCl₃):13.7 (CH₂CO₂CH₂CH₃); 38.4 (CH₂CO₂CH₂CH₃); 61.5 (CH₂CO₂CH₂CH₃); 134.3(C═C); 166.9 (CO₂); 169.6 (C═O). Anal. (C₈H₉NO₄) C, H, N, S. TABLE 4bThis lists the activity of further compounds of Family G in Table 4above, ie those compounds of formula:

wherein R is as listed in the table. Compound No. R GSK-3β IC₅₀ (μM) 7-GNH₂ >100 8-G CH₂(4-MeO—Ph) 65

[0351] GSK-3 inhibitors: For compounds belonging to family D, the GSK-3inhibition experiments were also performed at variable concentrations ofATP (up to 50 μM) and in all cases the same value of IC₅₀ were obtained.Thus could suggest that these compounds do not compete with ATP in thebinding to GSK-3.

Example 5 Cell Cycle Blockade

[0352] The IC₅₀ for some of the compounds tested in N₂A cell culturesare gathered in Table 5 below.

TABLE 5 R^(a) R^(b) X Y IC₅₀ (μM) CH₂Ph Me O O 4-8 Et Me O O  40-100 EtnPr O O  5-10 Et cyclohexyl O O 6-9 Ph Me O O 4-7 CH₂CO₂Et Me O O 1-24-OMePh Me O O 1-2 CH₂Ph Et O O 4-7 CH₂Ph CH₂Ph O O 2-3 Et Et O O 30-80CH₂Ph CH₂Ph O S 1-2 Ph Ph O S 4-8

1. Compounds of general formula (I):

wherein: A is —C(R¹)₂—, —O— or —NR¹—; E is —NR¹— or —CR¹R²— and thesubstituent R² is absent if - - - is a second bond between E and G; G is—S—, —NR¹— or —CR¹R²— and the substituent R² is absent if - - - is asecond bond between E and G; - - - may be a second bond between E and Gwhere the nature of E and G permits and E with G optionally then forms afused aryl group; R¹ and R² are independently selected from hydrogen,alkyl, cycloalkyl, haloalkyl, aryl, —(Z)_(n)-aryl, heteroaryl, —OR³,—C(O)R³, —C(O)OR³, —(Z)_(n)—C(O)OR³ and —S(O)_(t)— or as indicated R²can be such that E with G then form a fused aryl group; Z isindependently selected from —C(R³)(R⁴)—, —C(O)—, —O—, —C(═NR³)—,—S(O)_(t)— and N(R³)—; n is zero, one or two; t is zero, one or two; R³and R⁴ are independently selected from hydrogen, alkyl, aryl andheterocyclic; and X and Y are independently selected from ═O, ═S, ═N(R³)and ═C(R¹)(R²).
 2. A compound according to claim 1, wherein A isselected from —C(R¹)₂— and —NR¹—, and R¹ is defined in claim
 1. 3. Acompound according to claim 1, wherein R¹ and R² are independentlyselected from hydrogen, alkyl, cycloalkyl, aryl (optionally substitutedwith a group selected from alkyl, halo and alkoxy), —C(R³)(R⁴)-aryl (thearyl part being optionally substituted with a group selected from alkyl,halo and alkoxy), —OR³, —C(O)OR³ and —C(R³)(R⁴)—C(O)OR³, and R³ and R⁴are independently selected from hydrogen and alkyl.
 4. A compoundaccording to claim 1, wherein X and Y are independently selected from ═Oand ═S.
 5. A compound according to claim 1, wherein: A is selected from—C(R¹)₂— and —NR¹—, and R¹ is selected from hydrogen, alkyl, cycloalkyl,aryl (optionally substituted with a group selected from alkyl, halo andalkoxy), —C(R³)(R⁴)-aryl (the aryl part being optionally substitutedwith a group selected from alkyl, halo and alkoxy), —OR₃, —C(O)OR³ and—C(R³)(R⁴)—C(O)OR³, and R³ and R⁴ are independently selected fromhydrogen and alkyl; and X and Y are independently selected from ═O and═S.
 6. A compound according to claim 1, having the general formula (II):

wherein: R^(a) and R^(b) are independently selected from hydrogen,alkyl, cycloalkyl, haloalkyl, aryl, —(Z)_(n)-aryl, heteroaryl, —OR³,—C(O)R³, —C(O)OR³, —(Z)_(n)—C(O)OR³ and —S(O)_(t)—, and Z, n, t, R³, R⁴,X and Y are as defined in claim
 1. 7. A compound according to claim 6,wherein R^(a) and R^(b) are independently selected from hydrogen, alkyl,cycloalkyl, aryl (optionally substituted with a group selected fromalkyl, halo and alkoxy), —C(R³)(R⁴)-aryl (the aryl part being optionallysubstituted with a group selected from alkyl, halo and alkoxy), —OR³,—C(O)OR³ and —C(R³)(R⁴)—C(O)OR³, and R³ and R⁴ are independentlyselected from hydrogen, alkyl and heterocyclic.
 8. A compound accordingto claim 7, wherein R^(a) and R^(b) are independently selected fromalkyl, aryl (optionally substituted with a group selected from alkyl,halo and alkoxy), —CH₂-aryl (the aryl part being optionally substitutedwith a group selected from alkyl, halo and alkoxy), and —CH₂—C(O)OR³,and R³ is hydrogen or alkyl.
 9. A compound according to claim 8, whereinR^(a) and R^(b) are independently selected from methyl, ethyl, propyl,benzyl, phenyl (optionally substituted with a group selected frommethyl, fluoro, chloro, bromo and methoxy) and —CH₂—C(O)O-ethyl.
 10. Acompound according to claim 6, wherein X and Y are independentlyselected from ═O, ═S and ═NR³ (wherein R³ is heterocyclic).
 11. Acompound according to claim 10, wherein X is ═O.
 12. A compoundaccording to claim 11, wherein X is ═O and Y is ═O.
 13. A compoundaccording to claim 6, wherein: R^(a) and R^(b) are independentlyselected from hydrogen, alkyl, cycloalkyl, aryl (optionally substitutedwith a group selected from alkyl, halo and alkoxy), —C(R³)(R⁴)-aryl (thearyl part being optionally substituted with a group selected from alkyl,halo and alkoxy), —OR³, —C(O)OR³ and —C(R³)(R⁴)—C(O)OR³, R³ and R⁴ areindependently selected from hydrogen, alkyl and heterocyclic, and X andY are independently selected from ═O, ═S and ═NR³.
 14. A compoundaccording to claim 13, wherein: R^(a) and R^(b) are independentlyselected from hydrogen, alkyl, cycloalkyl, aryl (optionally substitutedwith a group selected from alkyl, halo and alkoxy), —C(R³)(R⁴)-aryl (thearyl part being optionally substituted with a group selected from alkyl,halo and alkoxy), —OR³, —C(O)OR³ and —C(R³)(R⁴)—C(O)OR³, R³ and R⁴ areindependently selected from hydrogen and alkyl; and X is ═O.
 15. Acompound according to claim 14, wherein: R^(a) and R^(b) areindependently selected from methyl, ethyl, propyl, benzyl, phenyl(optionally substituted with a group selected from methyl, fluoro,chloro, bromo and methoxy) and —CH₂—C(O)O-ethyl; X is ═O; and Y is ═O.16. The following compounds according to claim 6: R^(a) R^(b) X Y CH₂PhMe O O Et Me O O Ph Me O O CH₂CO₂Et Me O O 4-OMePh Me O O 4-MePh Me O O4-BrPh Me O O 4-FPh Me O O 4-ClPh Me O O CH₂Ph CH₂Ph O S Ph Ph O S


17. A compound according to claim 1, having the general formula (III):

wherein: B is —NR⁷— or C(R⁷)(R⁸)— (wherein R⁷ and R⁸ are independentlyselected from hydrogen, alkyl, aryl, —CH₂-W-aryl, and —W—CO₂H, and W isa single bond, CH₂ or CO); R⁵ and R⁶ are independently selected fromhydrogen, alkyl, aryl and —CH₂-aryl; and X and Y are independentlyselected from ═O and ═S.
 18. A compound according to claim 17, wherein Bis —NR⁷—, and R⁷ is selected from hydrogen, alkyl and —CH₂-aryl.
 19. Acompound according to claim 18, wherein B is —NR⁷—, and R⁷ is hydrogen,methyl or benzyl.
 20. A compound according to claim 17, wherein R⁵ andR⁶ are hydrogen.
 21. A compound according to claim 17, wherein X is ═Oand Y is ═O.
 22. The following compounds according to claim 17: B X Y R⁵R⁶ NH O O H H N-CH₂Ph O O H H NMe O O H H CH₂ O O H H


23. A pharmaceutical formulation containing as active ingredient acompound as defined in any of claims 1 to
 22. 24. Use of a compound asdefined in any of claims 1 to 22 in the preparation of a pharmaceuticalformulation.
 25. Use according to claim 24, where the pharmaceuticalformulation is for the treatment of a disease in which GSK-3 isinvolved, including Alzheimer's disease or non-insulin dependentdiabetes mellitus.
 26. Use according to claim 24 where thepharmaceutical formulation is for the treatment of a hyperproliferativedisease such as cancer, displasias or metaplasias of tissue, psoriasis,arteriosclerosis or restenosis.
 27. A method for the treatment of adisease in which GSK-3 is involved, comprising administering to a humanin need of such treatment an effective amount of a compound according toany one of claims 1 to
 22. 28. A method according to claim 27, where thedisease is selected from Alzheimer's disease and non-insulin dependentdiabetes mellitus.
 29. A method according to claim 27, where the diseaseis a hyperproliferative disease such as cancer, displasias ormetaplasias of tissue, psoriasis, arteriosclerosis or restenosis.